Since the development of efficient and reliable methods for automated synthesis of oligonucleotides, and early observations about the potential therapeutic application of oligonucleotides, there is a high demand for new oligonucleotide analogues. This demand is due to the fact that natural oligonucleotides undergo very rapid nucleolytic degradation to monomeric nucleosides and nucleotides in biological fluids in vitro and/or in vivo.
The therapeutic application of oligonucleotides is based on the selective formation of hybrids between antisense oligonucleotides and complementary nucleic acids, such as messenger RNAs (mRNAs). Such hybrids inhibit gene expression by blocking protein translation. Successful inhibition of gene expression, however, requires the antisense oligonucleotide to be nuclease resistant so that it can be transported through biological membranes and can hybridize selectively to a target complementary nucleic acid, thereby actively blocking protein translation. Among the diverse oligonucleotide analogues that have been tested for antisense activity, those bearing phosphorothioate internucleotide linkages are the most nuclease resistant and, therefore, are the most widely used.
Oligonucleotides bearing phosphorothioate internucleotide linkages are typically prepared by sulfurization of a phosphite precursor which, in effect, substitutes a sulfur atom for one of the non-bridging oxygen atoms normally present in phosphodiesters. This substitution results in a stereogenic center at the phosphorus atom. Unfortunately, the sulfurization of oligonucleotide phosphodiesters prepared by conventional methods results in the formation of complex mixtures of diastereomers, since the precursors are typically diastereomeric with respect to phosphorus. The stereochemistry of the phosphorus center, however, is important in imparting nucleolytic stability in the oligonucleotide. Structural studies suggest that chirality at the phosphorus center alters the thermodynamics of duplex formation and the pharmacokinetic profiles of therapeutic oligonucleotides. Thus, synthetic methods and intermediates that enable one to control the stereochemistry of such thioated oligonucleotides and to prepare particular thioated oligonucleotides in high stereochemical purity are highly desired. Such methods and intermediates would enable one to optimize the nucleolytic stability of phosphorothioate oligonucleotides. It is even more desirable to develop synthetic methods and intermediates that enable one to control the stereochemistry of tricoordinated, as well as thioated, oligonucleotides and to prepare them in high stereochemical purity.
The most commonly used synthetic method for the synthesis of thioated oligonucleotides is the phosphoramidite method with stepwise sulfurization (see, e.g., U.S. Pat. Nos. 4,415,732, 4,668,777, 4,973,679, 4,845,205, and 5,525,719). This method uses tricoordinated phosphorus precursors that normally produce products containing a mixture of different thioated oligonucleotide stereoisomers. The lack of stereoselectivity in the phosphoramidite process is primarily due to the non-stereoselective and non-stereospecific acid-catalyzed nucleophilic substitution reaction, which is typically required to effect substitution. Even when diastereomerically pure P-chiral precursors are used, the coupling reaction proceeds with full epimerization at phosphorus.
Attempts have been made to control the stereochemistry of phosphorus in the synthesis of oligonucleotides. One attempt is a recently developed stereoselective method, which is drawn to the synthesis of thioated oligonucleotides using tricoordinated phosphorus precursors for acid-catalyzed nucleophilic substitution reactions. However, this approach has narrow applicability, in that it is limited to the synthesis of very short oligomers, particularly because each successive nucleoside coupling step occurs without complete stereoselectivity.
Another attempt at controlling phosphorus stereochemistry in oligonucleotide synthesis involves a method for the stereospecific synthesis of thioated oligonucleotides utilizing tetracoordinated phosphorus precursors to accommodate base-catalyzed nucleophilic substitutions. However, this approach also has limited applicability because a different type of tetracoordinated phosphorus precursor must be used to generate a particular type of product, for example, phosphates, phosphorothioates and phosphoroselenoates. In other words, the structure of the desired product is determined by the structure of the tetracoordinated phosphorus precursor at the coupling step. Additionally, separation of the diastereomers is difficult, and these tetracoordinated phosphorus precursors also are hydrolytically unstable.
In view of the foregoing problems, there exists a need for methods and intermediates that will permit the efficient synthesis of unmodified or modified oligonucleotides, particularly P-chiral oligonucleotides, with high stereospecificity. The present invention provides such methods and associated intermediates. These and other advantages of the present invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.